Retroviral vector plasmids
Lentiviral vector plasmids pRRL.PPT.EFS.EGFP.pre [50,51,52] and pRRL.PPT.SFFV.DsRedExpress.pre  were referred to as LV and LV.DsRed, respectively. Gammaretroviral vectors pSERS11.EFS.EGFP.pre  and pSF91.DsRedExpress  were termed GV and GV.DsRed. Except for the LTR-driven GV.DsRed, all vectors had SIN designs with deletions of the viral U3 regions (∆U3), instead containing the internal EFS promoter (short version/250 bp fragment of promoter/enhancer sequences from the human elongation factor 1 alpha gene) or, for LV.DsRed, the SFFV promoter (promoter/enhancer sequences from spleen focus forming virus). For reprogramming, the lentiviral “4-in-1” construct pRRL.PPT.SF.hOKSMco.id.Tom.pre.F+F3, co-expressing codon-optimized versions of the transcription factors Oct4, Klf4, Sox2 and wt c-Myc, was used [15, 54]. The construct contained heterospecific FRT sites (F and F3) to allow Flp recombinase-mediated excision of the reprogramming cassette [15, 55].
Cells and cultivation
Human 293T (embryonic kidney cell line) , human HT1080eCat (fibroblast cell line HT1080  expressing the murine ecotropic receptor mCAT-1 ) and the murine ECC line F9 (kindly provided by S. P. Goff, Columbia University Medical Center, New York, NY, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Biochrom GmbH, Berlin, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (GE Healthcare Europe GmbH, Freiburg, Germany), 100 U/mL penicillin, 100 µg/mL streptomycin and 1 mM sodium pyruvate (all from PAN-Biotech, Aidenbach, Germany). CF-1 Mefs (MTI Global Stem, Gaithersburg, MD, USA) and C3H Mefs (kindly provided by T. Cantz, Hannover Medical School, Hannover, Germany) as well as freshly isolated murine adult fibroblasts (C57BL/6 p14f/f) were grown in low glucose DMEM (PAN-Biotech) supplemented with 15% heat-inactivated FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM l-glutamine, 1% MEM non-essential amino acids solution (Gibco Life Technologies GmbH, Darmstadt, Germany) and 100 µM beta-mercaptoethanol (Sigma-Aldrich, Munich, Germany) on gelatin pre-coated (0.1% gelatin in phosphate buffered saline (PBS)) wells or flasks. PSC were co-cultured with irradiated (30 Gy) C3H Mefs in ESC medium composed of Knockout DMEM (Gibco Life Technologies) with 15% ESC-tested and heat-inactivated FBS (GE Healthcare Europe GmbH), 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 1% MEM non-essential amino acids, 100 µM beta-mercaptoethanol and 103 U/mL LIF (kindly provided by the Department of Technical Chemistry, Leibniz University Hannover, Hannover, Germany). The following murine PSC were used in this study: iPSC clone #1 (C57BL/6 mouse strain), clones #2, #2EX (excised reprogramming cassette) and #3 (C57BL/6 p14f/f, wt version without conditional knockout of the p14 gene, reprogrammed from adult fibroblasts, mice kindly provided by D. Kotlarz and C. Klein, Ludwig Maximilian University, Munich, Germany), clone #4 (reprogrammed from C3H Mefs) and ESC (C57BL/6 mouse strain, kindly provided by I. Prinz, Hannover Medical School).
Retroviral particle production
One day before transfection, 5 × 106 293T cells were seeded per 10-cm dish. Transfection was performed based on the calcium phosphate precipitation method assisted by 25 µM chloroquine (Sigma-Aldrich). For LV or LV.DsRed, packaging cells were co-transfected with 5 μg LV plasmid, 5 μg pRSV-Rev (kindly provided by T. Hope, Northwestern University, Chicago, IL, USA), 1.5 μg pMD.G (VSVg) , 12 μg wt pcDNA3.gp.4xCTE (HIV-1 Gag-Pol)  or CypA-independent capsid mutant Gag-Pol P90A, G89V, A88T or H2.1 or the N74D mutant [36, 38, 39, 41]. Mutations were introduced by overlap extension PCR and confirmed by sequencing. For production of LV-Ampho(tropic) and LV-Eco(tropic) particles, 2 μg ecotropic  or 2 μg amphotropic  MLV envelope encoding plasmids were used. To package VSVg pseudotyped GV or GV.DsRed particles, 5 μg GV plasmid, 7 μg pcDNA3.MLV.GP (MLV Gag-Pol)  and 1.5 μg pMD.G were co-transfected. Supernatants were harvested 36 and 48 h after transfection, filtered through 0.22 μm pore-size-filters and concentrated (100×) by ultracentrifugation (2 h, 82,740×g, 4 °C) (SW32Ti rotor; Beckman Coulter GmbH, Krefeld, Germany). Viral pellets were resuspended in ESC medium and stored in aliquots at −80 °C until further usage.
Retroviral particle titration and transduction
One day before titration of vector supernatants, 7 × 104 HT1080eCat cells were seeded per well of a 12-well plate. The next day, the culture medium was removed and serial dilutions of vector supernatants were added to the cells. Viral transduction of cells was performed in the presence of 4 µg/mL protamine sulfate and spin-inoculation (1 h, 863×g, 37 °C). Three days after transduction, the percentage of fluorescent cells was analyzed by flow cytometry. Vector particle titers were calculated only including samples with < 30% transduced cells to avoid multiple proviral integration events per cell and false titer estimation. Mefs (3 × 104 cells per well of a 12-well plate) or ECC (5 × 104 cells per well of a 12-well plate) were seeded the day before transduction. Specific amounts of vector particles per cell, referred to as MOI, were applied and cells were treated as described above for titration. PSC were transduced in single cell suspension and separated with 0.5% Trypsin-EDTA in PBS (Gibco Life Technologies GmbH) on the day of transduction. Afterwards, PSC were centrifuged (138×g, 5 min) and depleted from feeder cells. Depletion was performed by incubating the cells on a 15-cm plate (40 min, 37 °C, 5% CO2) to separate the faster attaching feeder from PSC. After applying vector supernatants at specific MOI to 3 × 104 cells (12-well plate) and adding 4 µg/mL protamine sulfate, a spin-inoculation step (1 h, 863×g, 37 °C) and an incubation step (1 h, 37 °C, 5% CO2) were performed, before PSC were added to feeder cells. The next day, PSC had attached to the feeder cell layer, and the virus-containing medium was replaced by fresh ESC medium. For small molecule supplemented transductions, cells were incubated 30 min prior to and during transduction with indicated concentrations of CSA (Sigma - Aldrich), MG132 (Calbiochem/Merck Millipore, Darmstadt, Germany), Nevirapine (Sigma-Aldrich) or Raltegravir (Santa Cruz Biotechnology, Dallas, TX, USA). Depending on the cell type and experiment, cells were processed by flow cytometry 2 days (differentiated cultures), 3 days (HT1080eCat titration, C3H Mefs), 5–8 days (adult fibroblasts, ESC, ECC, iPSC) or at indicated time points after transduction.
Reprogramming murine primary fibroblasts to iPSC
Murine fibroblasts from the indicated mouse strains were reprogrammed and characterized as previously described by our group [15, 54].
Differentiation of iPSC
To induce differentiation, iPSC were depleted from feeder cells, seeded in gelatin pre-coated (0.1% gelatin in PBS) 12-well plates (5 × 104 per well) and cultured in ESC medium without LIF and feeder cells. Medium was exchanged daily and cells were split in the range of 1:5–1:20 on days 3–4 and 5–6. Prior to transduction of differentiated cultures, cells were stained for SSEA1 expression and analyzed by flow cytometry to investigate their stem cell status. Differentiated cultures were transduced with retroviral particles (MOI 100) encoding EGFP or DsRedExpress 6–9 days after starting differentiation. Cells transduced with DsRedExpress encoding vectors were additionally stained with Stain Alive SSEA1 Antibody DyLight 488 according to the manufacturer’s protocol (Stemgent, Cambridge, MA, USA) and analyzed by fluorescence microscopy 2 days after transduction. Pictures were acquired with Axio Observer Z1 (Carl Zeiss AG, Jena, Germany) using Zeiss filter sets 43 (DsRedExpress) and 38 (DyLight 488) and AxioVision 4.8 software. Cells transduced with EGFP encoding vectors were analyzed by flow cytometry 2 days after transduction.
Cells were harvested and subsequently analyzed by flow cytometry (FACS Calibur, Becton-Dickinson, Heidelberg, Germany) using FlowJo software (Tree Star Inc, Ashland, OR, USA). In addition, PSC or differentiated cultures were stained with an Alexa 647 labeled SSEA1 antibody (1.5 ng per sample, 30 min, 4 °C) (eBioscience, San Diego, CA, USA). Samples were pre-gated for viable cells. Data from total viable cell populations with at least 50% SSEA1 positive cells were included in the analyses. The range of SSEA1 positive cells constituted mostly 70–100% throughout the study. The 70–100% range derives from differences between independent transduction experiments, but there were equivalent amounts of SSEA1 positive cells within each experiment. Cells, double positive for SSEA1 and EGFP, are shown in the graphs.
SYBR Green-based quantitative real-time PCR for detection of mean vector copy number per cell
iPSC were transduced with LV and GV and DNA was isolated 6–8 days after transduction. Vector copies per cell shown in Fig. 1c, e and Additional file 4B were determined by SYBR Green-based quantitative real-time PCR based on EGFP copies (Applied Biosystems, Darmstadt, Germany) using the QuantiTect SYBR-Green PCR Kit (Qiagen, Hilden, Germany) normalized to endogenous PTBP2 (EGFP for: 5′CTATATCATGGCCGACAAGCAGA3′, rev: 5′GGACTGGGTGCTCAGGTAGTGG3′; PTBP2 for: 5′GTCTCCATTCCCTATGTTCATGC3′, rev: 5′GTTCCCGCAGAATGGTGAGGTG3′). Master Mix preparation and PCR were performed as instructed by the manufacturer. Quantification of the mean vector copy number per cell were determined based on the comparative 2−∆∆Ct method and based on a reference plasmid containing EGFP and PTBP2 sequences [62,63,64].
Analysis of RT products, 2-LTR circles and proviral integration by TaqMan-based quantitative real-time PCR
Vector supernatants were applied (MOI 10) to iPSC and CF-1 Mefs and remaining vector particles were removed by washing the cells twice with PBS 6 h after transduction. Cells were harvested at indicated time points. To test for plasmid contamination of vector supernatants, equivalent volumes of vector supernatants were heat-inactivated at 65 °C for 1 h and included in the analyses (plasmid ctrl). DNA isolation was performed with the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s instructions. For TaqMan-based quantitative real-time PCR (Applied Biosystems), RT products were analyzed for early RT products (strong-stop DNA, for: 5′GCCTCAATAAAGCTTGCCTTGA3′, rev: 5′TGACTAAAAGGGTCTGAGGGATCT3′, probe: 5′AGAGTCACACAACAGACGGGCACACACTA3′), late RT products (U5/downstream PBS, for: 5′TAGTGTGTGCCCGTCTGTTG3′, rev: 5′GAGTCCTGCGTCGAGAGAG3′, probe: 5′TCCCTCAGACCCTTTTAGTCA3′) and 2-LTR circles (junction, for: 5′TAGTGTGTGCCCGTCTGTTG3′, rev: 5′CAGAGAGACCCAGTACAAGC3′, probe: 5′CTCTAGCAGTAACTGGAAGGGCT3′). PTBP2 served as a housekeeping control (for: 5′TCTCCATTCCCTATGTTCATGC3′, rev: 5′GTTCCCGCAGAATGGTGAGGTG3′, probe: 5′ATGTTCCTCGGACCAACTTG3′). RT products were relatively quantified using the comparative 2−∆∆Ct method [62, 63]. PCR was performed at 50 °C for 2 min and at 95 °C for 20 s followed by 60 cycles of 5 s at 95 °C, 20 s at 56 °C and 20 s at 65 °C.
B1-LTR PCR for analysis of proviral integration
Briefly, a first stage PCR was run with a LTR forward primer, which was designed to harbor a lambda-phage heel sequence at the 5′ end and LTR sequences at the 3′ end. In addition, two outward facing primers binding the highly redundant consensus sequence within the mouse B1 repetitive element were used in the first stage PCR. This was followed by a second stage TaqMan-based quantitative real-time PCR, which amplified the LTR using a lambda-specific forward and an LTR-specific reverse primer, scored using an LTR-specific probe. Primer sequences and protocol were used from Tervo et al. . For data analyses, genomic DNA from a SC-1 standard cell line, harboring three HIV-1-based vector integrations , was diluted over a range of concentrations covering three logs. Slope and y-intercept of the standard curve were used to determine proviral copies. PTBP2 served as a housekeeping control. To exclude plasmid contamination of vector supernatants, 5 µM Nevirapine was used as an RT inhibitor, for every time point with equivalent volumes of vector supernatants, and signals were subtracted from the total signal. For each sample, PCR controls, omitting the B1 forward and reverse primers or the Lambda-LTR forward primer, were performed in parallel during the first PCR stage. The approach omitting the Lambda-LTR forward primer did not reveal any signal. The signal achieved by omission of B1 forward and reverse primers was subtracted from the total signal for each sample.
Western blot analysis
Cells were harvested, washed with PBS and lysed by using radioimmunoprecipitation assay buffer supplemented with proteinase inhibitors (Complete Mini, Roche, Mannheim, Germany). Samples (15 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5%) and blotted onto nitrocellulose membranes (GE Healthcare Europe GmbH). Antibody probing was conducted with rabbit monoclonal anti-CypA (Cell Signaling Technology, Danvers, MA, USA) and rabbit polyclonal Erk2 (Santa Cruz Biotechnology) according to the manufacturer’s instructions. Goat anti-rabbit IgG conjugated with horseradish peroxidase was used as a secondary antibody. Quantitative detection was carried out using the Fusion Fx system (Peqlab GmbH/VWR Life Science Competence Center, Erlangen, Germany).
iPSC were pre-transduced with LV.DsRed or GV.DsRed at an MOI of 1000. After spin-inoculation cells were incubated at 37 °C and 5% CO2. After 6 h incubation time, iPSC were washed twice with PBS and a second transduction with LV was performed. LV encoding EGFP at an MOI of 100 were used for the second transduction. Flow cytometry analyses were conducted 4 days after transduction experiments.
Data were expressed as mean ± standard deviation. Where appropriate, we used one-way ANOVA with Tukey-Kramer post hoc test to adjust for multiplicity effects. When comparing all groups to one control group, we utilized one-way ANOVA with Dunnett post hoc test. For time course analyses, we utilized repeated measures one-way ANOVA with appropriate post hoc test. The unpaired t test was performed for comparison of two groups. In case of significantly different variances between the groups, the unpaired t test with Welch’s correction was applied. p values of ≤ 0.05 were considered significant (*), ≤ 0.01 very significant (**), ≤ 0.001 extremely significant (***), and ns was considered not significant. Supplementary material and methods are described in Additional file 6.